High-Throughput Screening of G Protein-Coupled Receptor Antagonists Using a Bioluminescence Resonance Energy Transfer 1-Based β-Arrestin2 Recruitment Assay
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In this study, the authors developed HEK293 cell lines that stably coexpressed optimal amounts of beta-arrestin2-Rluc and VENUS fusions of G protein-coupled receptors (GPCRs) belonging to both class A and class B receptors, which include receptors that interact transiently or stably with beta-arrestins. This allowed the use of a bioluminescence resonance energy transfer (BRET) 1- beta-arrestin2 translocation assay to quantify receptor activation or inhibition. One of the developed cell lines coexpressing CCR5-VENUS and beta-arrestin2- Renilla luciferase was then used for high-throughput screening (HTS) for antagonists of the chemokine receptor CCR5, the primary co-receptor for HIV. A total of 26,000 compounds were screened for inhibition of the agonist-promoted beta-arrestin2 recruitment to CCR5, and 12 compounds were found to specifically inhibit the agonist-induced beta-arrestin2 recruitment to CCR5. Three of the potential hits were further tested using other functional assays, and their abilities to inhibit CCR5 agonist-promoted signaling were confirmed. This is the 1st study describing a BRET1-beta-arrestin recruitment assay in stable mammalian cells and its successful application in HTS for GPCRs antagonists.
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- Authors
- Hamdan, F. F., Audet, M., Garneau, P., Pelletier, J., & Bouvier, M.
- Affiliations
- Université de Montréal (CA)
- McGill University (CA)
- Venue
- SLAS Discovery
- Publisher
- Elsevier BV
- Published
- 2005-08
- Volume
- 10
- Issue
- 5
- Pages
- 463-475
- ISSN
- 2472-5552 (ISSN-L)
- OpenAlex
- W2094714066
- Cited by
- 183
- References
- 60
- Citations by year
- 2026: 2 · 2025: 4 · 2024: 2 · 2023: 3 · 2022: 4 · 2021: 9 · 2020: 8 · 2019: 6 · 2018: 8 · 2017: 5 · 2016: 6 · 2015: 17 · 2014: 8 · 2013: 8 · 2012: 16
- FWCI
- 4.64
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- SLAS DISCOVERY (publishedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Receptor Mechanisms and Signaling
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › bioluminescence and chemiluminescence research
- Health Sciences › Medicine › Oncology › Chemokine receptors and signaling
- Keywords
- G protein-coupled receptors, β-arrestin2, CCR5, chemokine receptor antagonists, high-throughput screening, HIV co-receptor, HEK293 cells, receptor antagonists
- MeSH terms
- beta-Arrestins, Animals, Automation, Calcium, Cell Line, Dose-Response Relationship, Drug, Drug Evaluation, Preclinical, Energy Transfer, HIV, Humans, Luminescent Measurements, Microscopy, Fluorescence, Plasmids, Time Factors, Genes, Reporter, Arrestins, Receptors, CCR5, Protein Transport, Receptors, G-Protein-Coupled, Macromolecular Substances, Renilla, Luciferases, Renilla, Green Fluorescent Proteins
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